Abstract Biodesy has developed the Biodesy Delta device, a platform based on second harmonic generation (SHG) that allows one to measure changes in biomolecule structure upon ligand binding in real-time and in a high- throughput manner (using multi well plates). The Biodesy Delta will be used to develop a novel, high- throughput, SHG-based, commercial screening platform for identifying new binders and studying known ligands that directly modulate the structure and function of G-protein coupled receptors (GPCRs). SHG is a non-linear optical process that is highly sensitive to orientational changes of a SH-active, 370Da dye probe, covalently attached to a protein of interest, and requires very small amount of protein to get a reading. Using SHG, Biodesy has measured a range of molecular interactions between target proteins and small molecules, fragments, peptides and antibodies. To enable the study of GPCRs, we have successfully developed a prototype surface that allows tethering proteins and is resistant to detergents used to solubilize and stabilize membrane protein samples in solution. The new plate surface demonstrated minimal non-specific binding of the purified, labeled, thermostabilized, neurotensin receptor 1 (NTR1) GPCR. We have also demonstrated that SHG dye labeled NTR1 tethered to the surface remains functional by examining the binding of two control ligands to the tethered protein. In this project, we will develop a SHG assay that can be utilized for GPCRs that have not been stabilized by mutagenesis, and are relevant targets for drug discovery efforts or require a more in-depth understanding of their function. The Adenosine A2A Receptor (A2AR) will be the model GPCR, due to the large amount of structural, biophysical and biochemical data, functional assays and a non-stabilized construct that we can use for our SHG assay validation. First, we will design and test for activity with the Biodesy Delta multiple A2AR constructs that differ in the length of the C-terminal tail, which is very flexible and can interfere with binding to the surface. This will allow us to choose the optimal A2AR that retains full activity after labeling for SHG and attachment to the well surface. We will then create a version of our assay that can utilize 1536-well plates for more efficient screening of chemical libraries. Finally, we will test our assay in a proof of concept screen of a library that contains 20,000 small molecules.